This research project encompasses a number of different approaches to both understand how current antitubercular chemotherapy works using the most modern technologies and to use this information to develop new and improved therapies and therapeutic approaches.[unreadable] [unreadable] Individual projects within this framework are; (1) understanding the activity of various drugs in animal models of tuberculosis therapy, (2) correlating responses seen in animal models of disease with the pathology and response to therapy observed in human TB patients, and (3) developing structural and functional imaging techniques using CT/PET for use in live, infected animals. [unreadable] [unreadable] One important aspect of the project relies on the development of advanced animal models for predicting drug efficacy under conditions that exactly mimic those experienced by TB patients. In partnership with scientists at the University of Pittsburg section scientists have been exploring the microenvironment of tuberculosis in both rabbits and non-human primates infected with MTb. Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe TM) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in three animal models of disease: Guinea pig, rabbit and non-human primate. Following infusion of pimonidazole into animals with established infections, lung tissues from all three showed discrete areas of pimonidazole-adduct formation surrounding necrotic and caseous regions of pulmonary granulomata by immunohistochemical staining. This labeling could be substantially reduced by housing the animal in an atmosphere of 95% O2. Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber-optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3 mm) pulmonary lesions were severely hypoxic (1.6 0.7 mmHg). Finally, metronidazole (Mtz), which has potent bacteriocidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total lung bacterial burden in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.[unreadable] [unreadable] NIH purchased and temporarily installed the Ceretom CT and the Siemens MicroPET Focus 220 into the NIH Division of Veterinary Research (DVR) surgery facility in late February 2008. These units and the radiation handling procedures were tested with uninfected rabbits for the next two months until personnel were comfortable with the operation of the machinery and the manipulation of the 18F-FDG. Several CT scans using this protocol were paired with a BioVet physiological monitoring equipment and PET imaging with 18F-FDG. After this burn-in the machines were moved into the ABSL-3 facility in Building 33. Final commissioning and permissions for operating the scanners was obtained in July 2008 and scanning of infected animals has begun.